摘要
Background:Aberrant DNA methylation patterns have been observed in neurodegenerative diseases,including Alz-heimer’s disease(AD),and dynamic changes in DNA methylation are closely associated with the onset and progres-sion of these diseases.Particularly,hypomethylation of the amyloid precursor protein gene(APP)has been reported in patients with AD.Methods:In this study,we used catalytically inactivated Cas9(dCas9)fused with Dnmt3a for targeted DNA methyla-tion of APP,and showed that the CRISPR/dCas9-Dnmt3a-mediated DNA methylation system could efficiently induce targeted DNA methylation of APP both in vivo and in vitro.Results:We hypothesized that the targeted methylation of the APP promoter might rescue AD-related neuronal cell death by reducing APP mRNA expression.The cultured APP-KI mouse primary neurons exhibited an altered DNA-methylation pattern on the APP promoter after dCas9-Dnmt3a treatment.Likewise,the APP mRNA level was significantly reduced in the dCas9-Dnmt3a-treated wild-type and APP-KI mouse primary neurons.We also observed decreased amyloid-beta(Aβ)peptide level and Aβ42/40 ratio in the dCas9-Dnmt3a-treated APP-KI mouse neurons compared to the control APP-KI mouse neurons.In addition,neuronal cell death was significantly decreased in the dCas9-Dnmt3a-treated APP-KI mouse neurons.Furthermore,the in vivo methylation of APP in the brain via dCas9-Dnmt3a treatment altered Aβplaques and attenuated cognitive and behavioral impairments in the APP-KI mouse model.Conclusions:These results suggest that the targeted methylation of APP via dCas9-Dnmt3a treatment can be a potential therapeutic strategy for AD.
基金
Ministry of Science and ICT,and Ministry of Health and Welfare(2021M3E5E5096464,Republic of Korea)
Basic Science Research Program of the National Research Foundation of Korea(NRF)funded by the Ministry of Education(NRF-2022R1A6A1A03053343).
